Vaccine for Modulating Between T1 and T2 Immune Responses

ABSTRACT

The present invention provides liposomal vaccines containing immunogenic lipopeptides that are capable of modulating the humoral and cellular immune responses in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.10/106,876, filed Mar. 27, 2002, which claims priority to U.S.provisional application No. 60/278,698 filed on Mar. 27, 2001, all ofwhich are herein incorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Apr. 30, 2012, is named34395-812-302-Sequence-Listing.txt and is 8 Kilobytes in size.

FIELD OF THE INVENTION

The present invention provides liposomal vaccines capable of modulatingthe immune response in vivo, particularly the humoral and cellularimmune responses.

BACKGROUND OF THE INVENTION

To effectively combat disease, a vaccine should ideally stimulateseveral immunological reactions, such as the production of antibodies(humoral immunity) and the mobilization of immunological cells (cellularimmunity).

A cellular immune response brings about a proliferation and stimulationof T-lymphocytes, such as cytotoxic (CTL) and delayed-typehypersensitivity (DTH) T-cells, which go on to activate macrophages andimpede the propagation of pathogens. The induction of a humoral responsecauses the body's B-cells to produce antibodies against the offendingpathogen. However, some intracellular pathogens and retroviruses surviveand are extremely resistant to humoral-based immune responses andrequire the stimulation of cytotoxic T-cells to destroy such biologicalinvaders.

Synthetic peptides are often used as antigenic epitopes and can betailor-made using standard peptide synthesis technologies so that theyinduce minimal side effects. However, such peptides typically invoke arelatively weak immunogenic response.

Nonetheless, immunogenicity can be boosted by attaching a lipid to thesynthetic peptide. It has been shown, for example, that lipidation ofsynthetic peptide antigens leads to the induction of strong T-cellproliferation, CTL, and antibody responses in immunized mice,chimpanzees, or humans (BenMohamed et al., Vaccine, 18, 2843-2855(2000); Gahery-Segard et al., J. Virology., 74, 1694-1703 (2000); Sethet al., AIDS Res. Hum. Retroviruses, 16, 337-343 (2000); Tsunoda et al.,Vaccine, 17, 675-685 (1999); BenMohamed et al., Eur. J. Immunol., 27,1242-1253 (1997); Vitiello et al., J. Clin. Invest., 95, 341-349(1995)).

A preparation of such antigens may be delivered in vivo using a vaccine“carrier,” but the carrier itself can become the target of the host'shumoral immune response. Thus, the host's antibodies act against thevaccine carrier and not the antigenic epitope, which can result in rapidclearance of the vaccine by anti-carrier antibodies, negating theusefulness of the actual vaccine.

The incorporation of the lipid moiety of a lipopeptide into a liposome,however, proves an extremely useful way in which to deliver an antigenin vivo without eliciting an immune response against the carrier.However, none of these advances assist in the modulation of one immuneresponse to another. That is, it has not previously been shown that aliposomally-bound lipopeptide can elicit cellular and humoral immuneresponses by altering the number of lipids attached to a single peptide.

The present invention, however, provides a novel way of invoking andmodulating between cellular and humoral immune responses by using asingle antigenic peptide and a carrier that does not stimulate humoralresponses against itself.

SUMMARY OF THE INVENTION

The invention is directed to a formulation of liposomes containingimmunogenic lipopeptides. The present invention is further directed tothe administration of such liposomes in formulations that are capable ofboth invoking and modulating an immune response in an individual.

In one embodiment, the invention provides a method for producingimmunogenic liposomes comprised of self assembling lipids, includinglipid-derivatives of immunogenic peptides. The liposomal formulation cancontain and deliver either monolipopeptides, dilipopeptides, or mixturesof each to invoke and, thereby, modulate a cellular, humoral, orcellular and humoral immune responses, respectively. The peptide is animmunogenic sequence of amino acids, representing an epitope or asimilar feature that is antigenic in nature.

Another embodiment of the invention provides a composition that canstimulate and modulate an immune response. Such a composition comprisesa liposomal vesicle, wherein the lipid bilayer of the liposomal vesiclecomprises at least one immunogenic monolipopeptide and at least onedilipopeptide, wherein the percentage of the monolipopeptide varies frommore than about 0 to less than about 100% and wherein the percentage ofthe dilipopeptide varies from more than about 0 to less than about 100%.

Yet another embodiment of the invention provides a method of stimulatinga cellular immune response comprises administering to a patient aneffective amount of at least one monolipopeptide and at least onedilipopeptide, wherein the monolipopeptide and the dilipopeptide areassociated with the same or different liposomal vesicle. In oneembodiment, the percentage of the monolipopeptide administered is morethan about 50%. The percentage of the monolipopeptide can also be morethan about 70% or more than about 90%.

Yet another aspect of the invention provides a method of stimulating ahumoral immune response comprising administering to a patient aneffective amount of at least one monolipopeptide and at least onedilipopeptide, wherein the monolipopeptide and the dilipopeptide areassociated with the same or different liposomal vesicle. In oneembodiment, the percentage of the monolipopeptide administered is morethan about 50%. The percentage of the monolipopeptide can also be morethan about 70% or more than about 90%.

The present invention also encompasses compositions wherein the peptideportion of the monolipopeptide or the dilipopeptide is derived from aprotein associated with a disease selected from the group consisting oftuberculosis, hepatitis B, malaria, and cancer. In a preferredembodiment, the peptide comprises at least 5 contiguous amino acids ofan immunogenic region of the protein. In a more preferred embodiment,the monolipopeptide or the dilipopeptide is designed from a MUC-1protein sequence. In an even more preferred embodiment, the MUC-1lipopeptide comprises the sequence GVTSAPDTRPAPGSTA (residues 2 to 16 ofSEQ ID NO: 1). In another preferred embodiment, the monolipopeptide orthe dilipopeptide is designed from a tuberculosis lipopeptide comprisingthe sequence DQVHFQPLPPAVVKLSDALIK (SEQ ID NO: 2). In another preferredembodiment, an antigenic MUC-1 peptide of the present invention can beselected from any part of the sequenceSGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVSSL (SEQ ID NO. 1).

In another embodiment, the monolipopeptide or the dilipopeptide isdesigned from a tuberculosis peptide. In a more preferred embodiment,the tuberculosis peptide has the sequence, DQVHFQPLPPAVVKLSDALIK (SEQ IDNO. 2).

In yet another embodiment, the instant invention uses a lipidatedantigenic hepatitis B peptide. In a preferred embodiment, the hepatitisB peptide has the sequence, IRTPPAYRPPNAPILK (SEQ ID NO. 3). In anotherpreferred embodiment, a malaria peptide may be modified to containlipids. In one preferred embodiment, the malaria peptide has thesequence, VTHESYQELVKKLEALEDAVK (SEQ ID NO. 4).

A method of stimulating both a cellular and humoral immune responsecomprising administering to a patient an effective amount of at leastone monolipopeptide and at least one dilipopeptide is also provided.Another aspect of the present invention provides a method of modulatingbetween a cellular and humoral immune response, comprisingsimultaneously administering a formulation comprising aliposomally-bound monolipopeptide and a formulation comprising aliposomally-bound dilipopeptide. Yet another aspect of the presentinvention provides a method of modulating between a cellular and humoralimmune response, comprising first administering a formulation comprisinga liposomally-bound monolipopeptide and then administering a formulationcomprising a liposomally-bound dilipopeptide. Yet another aspectprovides a method of modulating between a cellular and humoral immuneresponse, comprising first administering a formulation comprising aliposomally-bound dilipopeptide and then administering a formulationcomprising a liposomally-bound monolipopeptide. A method of stimulatinga cellular and humoral immune response, comprising administering aneffective amount of a formulation comprising a liposomally-bounddilipopeptide is also provided.

Both the foregoing general description and the following briefdescription of the drawing and the detailed description are exemplaryand explanatory and are intended to provide further explanation of theinvention as claimed. Other objects, advantages, and novel features willbe readily apparent to those skilled in the art from the followingdetailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows T cell proliferation and IFN-g levels in C57BL/6 miceimmunized two times with MUC1 Dilipo- or Monolipopeptides in liposomes(Protocol I346). FIG. 1 shows C57BL/6 mice were immunized subcutaneouslytwo times with a standard BLP25 liposomal vaccine, or with 16aa MUC1based Dilipo- or Monolipopeptide liposomal vaccines. Nine days after asecond immunization procedure a T cell proliferation and IFN-g levelswere tested in standard assays.

DETAILED DESCRIPTION OF THE INVENTION

An unexpected and surprising finding of the present invention is thatthe induction of immune responses in vivo can be modulated byadministering serially, or in combination, liposomally-bound peptidescomprising one lipid chain and liposomally-bound peptides comprising atleast two lipid chains.

The term “modulate” means some change, adjustment, or adaptation to acertain proportion. Thus, an immune response may be modulated bystimulating factors which bring into play a change in immunologicalactivity. The intensity of an immune response may also be modulated. Forexample, the intensity or level of T-cell proliferation can be madegreater or lower after administration of factors that effect modulation.In the present invention, those factors can be liposomally-boundlipopeptides.

It was surprisingly discovered that a peptide having one lipid chain mayinvoke a large cellular immune response (i.e., a “T1” immune response)when incorporated into a liposomal formulation, but only a minimal or nohumoral response. However, that same peptide, when incorporated into aliposome, can be made to induce a large humoral response (i.e., a “T2”response), accompanied by either a minimal or massive cellular response,when two lipids are attached to its surface. The range of cellularresponse observed with a lipopeptide varies depending upon the identityof the lipopeptide. The presence of one, two, or more than two lipids onan antigenic peptide may also increase or reduce the intensity by whichan immune response is activated. The intensity of an immune response andthe type of immune response that is stimulated can also be modulated byvarying the number of amino acid residues between two lipids on anantigenic peptide.

The present invention demonstrates that attaching more than one lipidchain to a peptide bound to a liposomal bilayer stimulates a largelyhumoral immune response, along with variable cellular activity rangingfrom minimal to massive. The present invention also shows that attachinga second lipid to an antigenic peptide increases the level of T-cellproliferation as well as inducing antibody production as compared to themonolipid derivative. The term “derivative” means a compound derived orobtained from another that contains essential elements of the parentsubstance. Thus, the lipid derivative of an antigen refers to a peptidethat has at least one lipid attached. Hence it is possible to administeran effective amount of a liposomal formulation comprising onlydilipopeptides to invoke antibody production and cellular activity. An“effective amount” of liposomally-bound lipopeptide formulation, refersto an empirically-derived amount of that lipopeptide that modulates animmune response.

It was also discovered that for some antigens a dilipopeptide lipsomalformulation will trigger a massive cellular response, in addition to alarge humoral response.

Specifically, the examples show that liposomal formulations having amonolipopeptide induced largely cellular responses and minimal humoralresponses, and liposomal formulations having a dilipopeptide inducedlargely humoral responses and minimal cellular responses, for MUC-1,tuberculosis, and hepatitis B peptides. Administering a combination of adi- and a monolipopeptide resulted in superior cellular and humoralresponses.

Another advantage of the present invention is that differentlipopeptides can be incorporated into a liposome and thus be transportedand presented to the immune system simultaneously and under the sameconditions. Thus, it is possible to induce in an individual multipleimmune responses by treating that individual with a liposome thatcontains a T1-inducing monolipopeptide and a T2-inducing dilipopeptide.Alternatively, a mixture of liposomally-bound monolipopeptides andliposomal-bound dilipopeptides can also be used to simultaneously inducemultiple immune responses.

Thus, the present invention provides novel compositions and methods formodulating an immune response by varying the number of lipids that areattached to an antigenic peptide. An immune response can be modulated byadding a two or more lipids to an antigenic peptide, as can theintensity of the immune response. Furthermore, the intensity of animmune response can be modulated by varying the spacing of amino acidsbetween lipids.

An immune response can also be invoked by injecting pre-stimulatedantigen-presenting cells or T-cells into a patient. Known as “adoptiveimmunotherapy,” this technique creates in vitro an expanded populationof antigen-specific cells that are primed to combat the causative agentonce reintroduced into the body. In essence, cells are removed from thepatient, stimulated in vitro and reinjected back into the patient'sbloodstream. Specifically, peripheral blood lymphocytes, such as antigenpresenting cells, are isolated and then “charged” by exposing the cellsto antigens in vitro. The antigen becomes endocytosed by an antigenpresenting cell, whereupon it becomes associated with a majorhistocompatibility complex and subsequently presented on the outersurface of the cell. This population of primed cells can then bereinjected into the patient. Fractionating the peripheral bloodlymphocytes into dendritic cells and/or macrophages prior to chargingcan also be performed.

Thus, a liposomal formulation of the present invention, comprisingmembrane-bound, antigenic lipopeptides can be added to the isolatedantigen presenting cells in vitro to “charge” them. For example, aliposomal formulation comprising a monolipo-MUC I peptide, a dilipo-MUCI peptide, or a combination of both peptides, can be used to chargeperipheral blood lymphocytes which are then reinjected into the patientas a cellular vaccine.

Alternatively, “adoptive T-cell transfer therapy” can be performed. Thisentails incubating a patient's T-cells with pre-charged antigenpresenting cells in vitro. The T-cells become activated and arere-administered to a patient suffering from, for example, anadenocarcinoma. For a description of art-recognized techniques foradoptive T-cell transfer therapy, see Bartels et al., Annals of SurgicalOncology, 3(1):67 (1996), incorporated by reference. Thus, according tothe present invention, a lipidated antigenic peptide is selected,incorporated into a liposome, and used to stimulate peripheral bloodlymphocytes, which can either be injected back into the patient or usedthemselves to activate isolated T-cells. A T-cell activation method isalso useful for generating cytotoxic and helper T-cell responses toantigens involved in various pathological conditions, such as cancer,tumors, viral infections, and bacterial infections.

Liposomes

Liposomes are microscopic vesicles that consist of one or more lipidbilayers surrounding aqueous compartments. See e.g., Bakker-Woudenberget al., Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1): S61 (1993)and Kim, Drugs, 46: 618 (1993). Because liposomes can be formulated withbulk lipid molecules that are also found in natural cellular membranes,liposomes generally can be administered safely and are biodegradable.

Depending on the method of preparation, liposomes may be unilamellar ormultilamellar, and can vary in size with diameters ranging from about0.02 μm to greater than about 10 μm. A variety of agents can beencapsulated in liposomes. Hydrophobic agents partition in the bilayersand hydrophilic agents partition within the inner aqueous space(s). Seee.g., Machy et al., LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY (JohnLibbey, 1987), and Ostro et al., American J. Hosp. Pharm. 46: 1576(1989).

Liposomes can adsorb to virtually any type of cell and then release anincorporated agent. Alternatively, the liposome can fuse with the targetcell, whereby the contents of the liposome empty into the target cell.Alternatively, a liposome may be endocytosed by cells that arephagocytic. Endocytosis is followed by intralysosomal degradation ofliposomal lipids and release of the encapsulated agents. Scherphof etal., Ann. N.Y. Acad. Sci., 446: 368 (1985).

Other suitable liposomes that are used in the methods of the inventioninclude multilamellar vesicles (MLV), oligolamellar vesicles (OLV),unilamellar vesicles (UV), small unilamellar vesicles (SUV),medium-sized unilamellar vesicles (MUV), large unilamellar vesicles(LUV), giant unilamellar vesicles (GUV), multivesicular vesicles (MVV),single or oligolamellar vesicles made by reverse-phase evaporationmethod (REV), multilamellar vesicles made by the reverse-phaseevaporation method (MLV-REV), stable plurilamellar vesicles (SPLV),frozen and thawed MLV (FATMLV), vesicles prepared by extrusion methods(VET), vesicles prepared by French press (FPV), vesicles prepared byfusion (FUV), dehydration-rehydration vesicles (DRV), and bubblesomes(BSV). The skilled artisan will recognize that the techniques forpreparing these liposomes are well known in the art. See COLLOIDAL DRUGDELIVERY SYSTEMS, vol. 66 (J. Kreuter, ed., Marcel Dekker, Inc., 1994).

Lipids

A “lipid” may be a myristyl, palmitoyl, or a lauryl molecule, that canbe attached to amino acids that possess functional oxygen, nitrogen, orsulfur groups. Such amino acids include, but are not limited to,threonine, serine, lysine, arginine, and cysteine amino acids. A“monolipopeptide” is a peptide to which only one lipid chain isattached. Similarly, a “dilipopeptide” is a peptide that has two lipidchains attached to one or two amino acids. If the two lipid chains areattached to two amino acid residues, those residues can be spaced anynumber of amino acids apart.

A “liposomal formulation” describes in vitro-created lipid vesicles inwhich mono- and/or dilipopeptides can be incorporated. Thus,“liposomally-bound” refers to a peptide that is partially incorporatedor attached to a liposome. A liposomal formulation may also be referredto as a “liposomal vaccine.” A liposomal formulation may comprise twotypes of liposomes; one that contains mostly, if not all,monolipopeptides incorporated into its structure, and a second thatcontains mostly, if not all, dilipopeptides in its structure. Individualpreparations of “mono-” and “di-” liposomes can be administered togetherto modulate an immune response, even though the monolipopeptide anddilipopeptide do not exist on one liposome.

When incorporated into a liposome, the monolipopeptide and thedilipopeptide may be peptides that are the same antigenic epitope.Alternatively, the peptide sequences for each lipopeptide may comprisedifferent epitopes. The lipopeptides may be antigens that are associatedwith the same or different proteins.

A lipopeptide can be incorporated into liposomes because the lipidportion of the peptidic molecule will spontaneously integrate into thelipid bilayer. Thus, a lipopeptide may be presented on the “surface” ofa liposome. Alternatively, a peptide may be encapsulated within aliposome. Techniques for preparing liposomes and formulating them withmolecules such as peptides are well known to the skilled artisan.

Exemplary Adjuvants

The present liposomal vaccines may also be formulated advantageouslywith an adjuvant. As conventionally known in the art, adjuvants aresubstances that act in conjunction with specific antigenic stimuli toenhance the specific response to the antigen. Monophosphoryl lipid A(MPLA), for example, is an effective adjuvant that causes increasedpresentation of liposomal antigen to specific T Lymphocytes. Alving, C.R., Immunobiol., 187.430-446 (1993). The skilled artisan will recognizethat lipid-based adjuvants, such as Lipid A and derivatives thereof, arealso suitable. A muramyl dipeptide (MDP), when incorporated intoliposomes, has also been shown to increase adjuvancity (Gupta R K etal., Adjuvants—A balance between toxicity and adjuvancity, “Vaccine, 11,293-306 (1993)).

Another class of adjuvants includes stimulatory cytokines, such as IL-2.Thus, the present liposomal vaccines may be formulated with IL-2, orIL-2 may be administered separately for optimal antigenic response. IL-2is beneficially formulated with liposomes.

Exemplary Vaccine Formulations

Vaccines may also be formulated with a pharmaceutically acceptableexcipient. Such excipients are well known in the art, but typicallyshould be physiologically tolerable and inert or enhancing with respectto the vaccine properties of the inventive compositions. Examplesinclude liquid vehicles such as sterile, physiological saline. Anexcipient may be added at any point in formulating a liposomal vaccineor it may be admixed with the completed vaccine composition.

Vaccines may be formulated for multiple routes of administration.Specifically preferred routes include intramuscular, subcutaneous, orintradermal injection, aerosol, or oral administration, or by acombination of these routes, administered at one time or in a pluralityof unit dosages. Administration of vaccines is well known and ultimatelywill depend upon the particular formulation and the judgement of theattending physician. Vaccine formulations can be maintained as asuspension or they may be lyophilized and hydrated later to generate auseable vaccine.

To provide greater specificity, thus reducing the risk of toxic or otherunwanted effects during in vivo administration, it is advantageous totarget the inventive compositions to the cells through which they aredesigned to act, namely antigen-presenting cells. This may convenientlybe accomplished using conventional targeting technology to direct aliposome containing an immunogenic peptide to a particular locationwithin the body. To target antigen presenting cells, for example,mannose and the Fc portion of antibodies can be chemically conjugated toan antigenic peptide, or by recombinantly fusing the targeting peptideto the immunogenic lipopeptide. Other, similar strategies will befamiliar to the practitioner.

Exemplary Quantities of Lipopeptides and Liposomal Formulations

The ratio of antigenic monolipopeptides and dilipopeptides within aliposome can be varied so as to modulate an immune response to differentdegrees of intensity. For example, increasing the amount ofdilipopeptide incorporated in a liposome relative to the amount ofmonolipopeptide may make the resulting formulation morehumoral-inducing. Of course, due to differing magnitudes of response todifferent antigens, different ratios may be needed to achieve thedesired balance of humoral and cellular response.

For example, the skilled artisan can create liposomes made up of a ratioof covalently linked, immunogenic monolipopeptides and dilipopeptides,wherein the percentage of the monolipopeptide varies from more thanabout 0 to less than about 100% of the liposome. Similarly, thedilipopeptide can be present in the liposomal membrane as a percentageof more than about 0 to less than about 100%. For example, a liposomecomprising 75% monolipopeptide and 25% dilipopeptide may generate alargely T1 immune response with some T2 activity. The present inventionprovides methods for creating a liposome comprising about 1 to about 30%monolipopeptide, about 30 to about 50% monolipopeptide, or about 50 toabout 99% monolipopeptide. Similarly, a liposome comprising about 1 toabout 30% dilipopeptide, about 30 to about 50% dilipopeptide, or about50 to about 99% dilipopeptide can also be created. Thus, the presentinvention enables the creation of liposomes containing, for example,monolipopeptide: dilipopeptide in ratios such as about 10%:about 90%,about 30%:about 70%, about 50%:about 50%, about 70%:about 30%, about90%:about 10% and about 99%:about 1%. Determining relative antigenicityis within the purview of one having ordinary skill in immunology.

An effective amount of a liposomal formulation containing at least onemonolipopeptide and at least one dilipopeptide can be administered to apatient, wherein the monolipopeptide and dilipopeptide are associatedwith either the same or different liposomal vesicle. Thus a singleliposomal formulation comprising a ratio or ratios of a monolipopeptideand a dilipopeptide can be administered to a patient to invoke a desiredimmune response; or alternatively, combinations of at least twoliposomal formulation comprising different ratios of a monolipopeptideand a dilipopeptide can be administered to a patient to invoke a similaror different immune response.

“Treating” in its various grammatical forms in relation to the presentinvention refers to preventing, curing, reversing, attenuating,alleviating, minimizing, suppressing, or halting the deleterious effectsof a disease state, disease progression, disease causative agent, orother abnormal condition.

Exemplary Immunogenic Peptides Useful in the Invention

Any peptidic antigen or epitope may be lipidated and incorporated into aliposome for the purposes of inducing or modulating immune responses invivo. One, two, or more than two lipids can be added to any part of apeptide. The skilled artisan will recognize that the antigenic peptideis selected based upon the type of disease affecting the individual. Forexample, a “MUC-1” antigen is useful for making antigen-specific T-cellsthat can be used in treating adenocarcinoma. Similarly, a tuberculosispeptide, hepatitis B peptide, or a malaria peptide all can be lipidatedaccording to the present invention and used to invoke cellular andhumoral immunity as desired to treat the specific diseases with whichthe peptides are associated. The present invention is not limited to theuse of MUC-1, tuberculosis peptides, hepatitis B peptides, or malariapeptides as liposomally-bound lipopeptidic vaccines.

A variety of immunogenic peptides can be lipidated and incorporated intoliposomal membranes to create a number of different immunogenic-specificvaccines. Described herein, for example, is the immunogenic effect oflipidation upon a mucin-derived peptide. MUC I mucins are macromolecularglycoproteins expressed in all epithelial cells of healthy individuals.The core peptidic sequence of MUC-1 comprises 20 amino acid residuesthat are repeated throughout the protein anywhere from 60 to 120 times.The repeated sequence, GVTSAPDTRPAPGSTAPPAH (SEQ ID NO: 19), has fivepotential sites for glycosylation (bolded S, serine and T, threonine)and an immunogenic “DTR” epitope (underlined).

Generally, carbohydrates are linked to one or more of the serine orthreonine residues as O-linked structures and when all five sites areglycosylated, the epitope is concealed. In cancer cells, however, theglycosylation step is prematurely terminated such that the resultantcarbohydrates are truncated. Consequently, the DTR epitope is exposedand the peptidic core sequence and the carbohydrates become immunogenic.Thus, this peptide sequence is one example of an epitope that may beused in the context of the present invention so as to induce andmodulate an immune response. For example, an antigenic MUC I peptide ofthe present invention can be selected from any part of the sequenceSGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVSSL (SEQ ID NO. 1) andlipidated so as to contain one, two, or more than two lipids.

The size of an immunogenic peptide is also subject to variation.Lipidated MUC-1 peptidic molecules ranging from 16 (1882 Daltons) to 40(5050 Daltons) amino acids in size, for example, invoke immune responsesas described in the present invention. However, such immunogenicpeptides are not limited by size and may be a portion or even all of thedesired immunogen. Typically, small peptide antigens are preferred, dueto ease of manufacture and greater specificity. Thus, SGVTSAPDTRPAPGSTA(residues 1 to 17 of SEQ ID NO: 1) and STAPPAHGVTSAPDTRPAPGSTAPP(residues 15 to 39 of SEQ ID NO.: 1) are smaller MUC-1 peptides that canbe lipidated according to the present invention. Accordingly, unlessthey are multimeric (i.e., multiple copies of the same epitope), mostantigens will comprise from about 9 to about 100 amino acids. Morespecifically, antigens that are about 9 to about 20, about 20 to about40, about 40 to about 60, about 60 to about 80, and about 80 to about100 amino acids in size can be lipidated and incorporated into liposomesas described. Fragments of protein antigens can be produced byrecombinant DNA techniques and assayed to identify particular epitopes.Preferably, small peptides are produced by in vitro synthetic methodsand assayed. Thus, any antigenic sequence in whole, or in part, may beused in a lipidated form according to the instant invention. By “whole,or in part” it is meant that either the entire antigenic peptide or somesmaller peptide derived from the larger may be lipidated. A “smallerpeptide” may be a fragment of a larger peptide antigen or may besynthesized recombinantly or chemically.

Other illustrative examples of peptides that can be synthesized,lipidated, and used in liposomally prepared vaccines include, but arenot limited to, peptides involved in tuberculosis, hepatitis B, malaria,and cancer diseases.

The tuberculosis peptide DQVHFQPLPPAVVKLSDALIK (SEQ ID NO. 2), whichoriginates from a 38 kDa secretory protein of Mycobacteriumtuberculosis, can be made with one or two lipids and formulated intoliposomal formulations described above.

Similarly, a hepatitis B antigenic peptide is represented by the aminoacid sequence IRTPPAYRPPNAPILK (SEQ ID NO. 3). Likewise, the malariapeptide VTHESYQELVKKLEALEDAVK (SEQ ID NO. 4) also can be formulated intoa liposomal vaccine.

An amino acid, such as threonine, serine, lysine, arginine, or cysteine,which occurs within the natural sequence of an antigenic peptide, may bea convenient site to which a lipid can be linked. Alternatively, any oneof these amino acids can be added to either end or within a peptidesequence so as to facilitate the linking of a lipid moiety. Thus, anantigenic peptide can be made to have two lysine residues at itscarboxyl terminus so as to facilitate the linking of two lipids. By“made” the present invention contemplates the use of conventionalpeptide synthesis methods to introduce one or more additional aminoacids to a peptide sequence. However, recombinant methods also can beemployed to design polynucleotides that encode the desired amino acidsequence. Thus, the present invention envisions the chemical andrecombinant synthesis of antigenic peptides that are amenable tolipidation.

Throughout the specification, any and all references to a publiclyavailable document, including a U.S. patent, are specificallyincorporated by reference.

The examples below are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may be used.

Example 1

The purpose of this example was to determine the T-cell proliferationresponse and the Anti-MUC-1 antibody levels in response toadministration of a MUC-1 antigen liposomal vaccine.

Summary of Procedures Used (i) Immunization

MUC1 based liposomal vaccines (“BLP25 liposomal vaccines”) at a dose of100 μg (250 μl) was injected subcutaneously, two, three, or four times,at biweekly intervals, into right and left inguinal regions (125 μl pereach site).

(ii) T-Cell Proliferation Assay

Nine days after the last immunization with BLP25 liposomal vaccine allmice were sacrificed and lymph nodes were surgically excised. Nylon woolpurified lymph node T-cells were then cultured with antigen presentingcells (APCs), which were obtained from the spleens of naïve mice of thesame strain and treated with mitomycin C. These mixed cultures werepulsed with MUC1 derived synthetic lipopeptide (BP1-148) and controlpeptide (BP1-72) for four days. After the fourth day, some supernatantswere collected for IFN-g assay, and the cultures were then pulsed with afresh medium that contained a tritium labelled thymidine. After afurther 18-20 hours the incorporation of DNA-incorporated tritium wascounted in a liquid scintillation counter.

(iii) IFN-Gamma Assay

IFN-γ levels in collected supernatants were determined by a specificELISA using a sandwich technique. Briefly, 96 well Maxisorp flat bottomplates (Nunc, Denmark) were coated with 50 ul of catcher monoclonalantibody R4.6A2 (Biomira, lot#IM98A20A) for 35 min at 37° C., 5% CO₂.The plates were then washed and incubated 45 minutes with test samplesand with positive standard cytokine sample (Pharmingen lot#M031554).

After two washes the second biotinylated antibody was added: XMG1.2(Biomira lot#BG98G02B). After washing, peroxidase-conjugatedstreptavidin (Jackson ImmunoResearch lot#42350) was added and againincubation was 30 minutes. After 5 washes, 100 μl of HRPO substratesolution: 1 μl of 30% H₂O₂ diluted in 10 mL of 1 mg/mL ABTS (Aldrichlot#01328ES) buffered with citric acid and Na₂HPO₄. 7H₂O was preparedimmediately before use and added to each well. The optimal density wasmeasured with Thermomax ELISA reader at 405 nm wavelength in kineticmode for 10 minutes. Cytokine levels in the test sample were determinedby comparison with reference standards.

(iv) Anti-MUC1 Antibody Levels

Microtiter 96 well plates were coated with BP1-151HSA conjugate (MUC1 24amino acid peptide conjugated to HSA), or with Blend C (Blend C is anatural human MUC1 mucin purified from ovarian cancer ascites). Serialdilutions of sera were incubated on the antigen coated plates at roomtemperature for 1 hr, after which the wells were thoroughly washed.Peroxidase-labeled goat anti-mouse IgG specific antibody was added andincubated at room temperature for 1 hr. Each plate was then washed andABTS substrate was added. After 15 min the absorbance at 405 nm wasmeasured on an ELISA reader.

Example 2 Mono- and Dilipidated MUC I Antigenic Peptides

The purpose of this example was to demonstrate that a liposomally-boundMUC I peptide with two lipid chains dramatically increases theproduction of anti-MUC I antibodies in immunized mice as compared to aMUC I peptide with one lipid.

MUC 1 peptides were chemically synthesized to contain one or two lipids.“BP1-217” (SEQ ID NO: 5) has two liposerine residues attached at thecarboxy terminus of the core peptidic sequence and “BP1-228” (SEQ ID NO:6) has only one liposerine attached to the carboxy terminus. Themonolipopeptide and dilipopeptide were separately incorporated intoliposomes and the resultant liposomal formulations were evaluated asvaccines.

BP1-217: GVTSAPDTRPAPGSTAS(myristyl)S(myristyl)L BP1-228:GVTSAPDTRPAPGSTAS(myristyl)L

After at least two subcutaneous immunizations of C57BL/6 mice with thedilipopeptide liposomal vaccine, BP1-217, induced the T2, humoralresponse, producing very high levels of anti-MUC I immunoglobulin G(IgG). In contrast, a cellular response with very low levels of IgGproduced was invoked in mice immunized two times with themonolipopeptide, BP1-228. For example, BP1-217 produced anti-MUC I IgGtiters in the range of 1/72,000 to 1/218,700 on BP1-151 HSA solid phaseand 1/100- 1/2700 titers on Blend C solid phase, whereas BP1-228produced only low titers of IgG antibodies on BP1-151 HSA solid phase,and no antibodies were detected on Blend C solid phase. See Table 1,below.

BP1-228 or BLP-25, as monolipopeptides, produced very low or no antibodylevels as compared to any MUC1 peptide with 2 lipid chains. Allformulations tested are liposomal and contain lipid A adjuvant.

Anywhere from 40-100 μg of MUC1 lipopeptide can be used perimmunization, although the present invention is not limited to theseamounts, which may also vary according to the specific peptide used. 5μg of MUC1 peptide into liposomes elicited a strong T1 immune response,but no antibody production. In clinical trials, a 1000 μg dose of BLP25injected into patients was found to be very effective in eliciting aspecific T cell proliferation.

It is also shown that quantitative rather than qualitative differencesbetween lipid chains are important in eliciting T1 or T2 responses. Thatis, a humoral immune response can be elicited regardless of the type oflipid chain attached to the peptide. For instance, a MUC-1 peptide,“BP1-132A” (SEQ ID NO:7), that has two palmitoyl lysine lipophilic aminoacid residues attached to two adjacent lysine residues was also shown toinduce humoral immunity. See Table 2.

BP1-132A:

TAPPAHGVTSAPDTRPAPGSTAPPK(palmitate)K(palmitate)G

TABLE 1 Protocol 1346 loG Antibody Titer Data Summary BP1-151-HSABlend C Infected Mouse Log₂ Antibody  Log₂ Antibody Material # TiterTiter Titer Titer Group #1 BLP16 Dilipo 1 17.7  1/218,700 11.4  1/2700400 μg/mL 2 17.7  1/218,700 <6.6 <1/100 BP1-217  3 16.2  1/72,900  9.8 1/900 200 μg/mL 4 16.2  1/72,900  9.8  1/900 lipid A 5 16.2  1/72,90011.4  1/2700 Group #2 BLP16 Monolipo 1 11.4  1/2700 <6.6 <1/100400 μg/mL 2  9.8  1/900 <6.6 <1/100 BP1-228 3  9.8  1/900 <6.6 <1/100200 μg/mL 4  9.8  1/900 <6.6 <1/100 lipid A  5 <6.6 <1/100 <6.6 <1/100Group #3 BLP25 1  6.6  1/100 <6.6 <1/100 400 μg/mL  2 <6.6 <1/100 <6.6<1/100 BP1-148 3  1/100 <6.6 <1/100 200 μ/mL 4 <6.6 <1/100  8.2  1/300lipid A  5 <6.6 <1/100 <6.6 <1/100 Group #4 Saline 1 <6.6 <1/100 <6.6<1/100 2 <6.6 <1/100 <6.6 <1/100 3 <6.6 <1/100 <6.6 <1/100 4  6.6  1/100<6.6 <1/100 5 <6.6 <1/100 <6.6 <1/100 C57BI/6 mice were immunized twotimes with liposomal formulation: BLP16 Dilipo containing MUC1 basedlipopeptide (BP1-217) and lipid A, or BLP16 Monolipo containing MUC1based lipopeptide (BP1-228) and lipid A, or BLP25 containing MUC1 basedlipopeptide (BP1-148) and lipid A BP1-217GVTSAPDTRPAPGSTAS(Myristyl)S(Myristyl)L (SEQ ID NO: 5) BP1-228GVTSAPDTRPAPGSTAS(Myristyl)L (SEQ ID NO: 6) BP1-228GVTSAPDTRPAPGSTAS(Myristyl)L (SEQ ID NO: 6)

Two immunizations of dilipo-MUC I peptide liposomes stimulates mostlythe T2, humoral, immune response, although there remains some cellularimmune system activity, as seen by T cell proliferation and IFN-γproduction. Nonetheless, the invention shows that liposomaladministration of a MUC I peptide with two lipid chains (e.g., eitherpalmitoyl lysine or myristyl serine lipid chains) attached provides adramatic increase in antibody production and stimulation of the humoralimmune system.

This accomplishment has not previously been observed in any mammalianmodel.

TABLE 2 Protocol 1350B (2× immunization) IgM and IgG Antibody Titer Data BP1-151-HSA Blend C Injected Mouse IgM IgG IgM IgGMaterial # Titer Titer Titer Titer BLP24 Dilipo 1  1/72,900  1/218,700 1/100  1/300 400 μg/mL  2  1/24,300  1/218,700  1/100  1/300 BP1-132200 μg/mL  3  1/8100  1/218,700  1/100  1/900 lipid A ×2  4  1/24,300 1/218,700  1/100  1/300 immunization BLP25 1 <6.6 <1/100  1/100 <1/100400 μg/mL  2  6.6  1/100 <1/100 <1/100 BP1-148 200 μg/mL  3  8.2  1/300 1/100 <1/100 lipid A ×2  4 <6.6 <1/100  1/100 <1/100 immunizationSaline 1 <6.6 <1/100 <1/100 <1/100 2 <6.6 <1/100 <1/100 <1/100 ×2  3<6.6 <1/100 <1/100 <1/100 immunization 4 <6.6 <1/100 <1/100 <1/100C57B1/6 mice were immunized two times with liposomal formulation:BLP24Dilipo, containing MUC1 based lipopeptide (BP1-132B) and lipid ABLP25 containing MUC1 based lipopeptide (BP1-148) and lipid A BP1-132B(SEQ ID NO: 9) TAPPAHGVTSAPDTRPAPGSTAPPK(Palmitoyl)K(Palmitoyl)L

To further characterize the observed phenomenon, MUC I transgenic micewere immunized with MUC I based liposomal formulations containing mono-or dilipopeptides. As shown in Table 3, a strong IgG antibody responsewas again observed upon immunization with the dilipopeptide (BP1-217),but only after four immunizations. This shows that C57B1/6 MUC Itransgenic mice which are tolerogenic for MUC I antigen need moreimmunizations than normal C57B1/6 mice to break this tolerance andinduce high levels of anti-MUC I IgG. Mice immunized with only themonolipopeptide (e.g., BP1-228), however, showed very low titers of IgG.

Thus, administration of a liposomal formulation comprising monolipo-MUCI peptide stimulates a cellular response. Immunization with a dilipo-MUCI peptide liposomal formulation induces a humoral response. The creationof a liposome comprising monolipo-MUC I and dilipo-MUC I invokes bothcellular and humoral immune responses upon immunization. Hence,modulation of the immune response can be achieved by selectivelyadministering a particular liposomal formulation in a certain order.

TABLE 3 Protocol 1347C - MUC1 Transgenic Mice IgG  Antibody Titer Data (4 Immunizations) BP1-151-HSA Blend C Injected MouseLog₂ Antibody Log₂ Antibody Material # Titer Titer Titer Titer Group # 1BLP16Dilipo 1 16.2  1/72,900 <6.6 <1/100 400 μg/mL  2 16.2  1/72,900 6.6  1/100 BP1-217 3 14.6  1/24,300 <6.6 <1/100 200 μg/mL  4 16.2 1/72,300 <6.6 <1/100 lipid A Group # 2 BLP16Monolipo 1  8.2  1/300  8.2 1/300 400 μg/mL  2  9.8  1/900 <6.6 <1/100 BP1-228 3  9.8  1/900  8.2 1/300 200 μg/mL  4 <6.6 <1/100 <6.6  <1/100  lipid A Group # 3 BLP25 111.4 1/2700 <6.6 <1/100 400 μg/mL  2 <6.6 <1/100 <6.6 <1/100 BP1-228 3 9.8  1/900 <6.6 <1/100 200 μg/mL  4 <6.6 <1/100 <6.6 <1/100 lipid AGroup # 4 Saline 1 <6.6 <1/100 <6.6 <1/100 2 <6.6 <1/100 <6.6 <1/100 3<6.6 <1/100 <6.6 <1/100 4 <6.6 <1/100 <6.6 <1/100 5 <6.6 <1/100 <6.6<1/100 C57BI/6 MUC1 transgenic mice were immunized four times withliposomal formulation: BLP16 Dilipo containing MUC1 based lipopeptide(BP1-217) and lipid A, or BLP16 Monolipo containing MUC1 basedlipopeptide (BP1-228) and lipid A, or BLP25 containing MUC1 basedlipopeptide (BP1-148) and lipid A BP1-217 (SEQ ID NO: 5)GVTSAPDTRPAPGSTAS(Myristyl)S(Myristyl)L BP1-228 (SEQ ID NO: 6)GVTSAPDTRPAPGSTAS(Myristyl)L BP1-148 (SEQ ID NO: 8)STAPPAHGVTSAPDTRPAPGSTAPP-Lys(Palmitoyl)

Example 3 Mono- and Dilipidated Tuberculosis Peptides

As was observed for MUC I monolipo- and dilipopeptides, immunizationwith dilipo-tuberculosis peptide dramatically increased antibodyproduction. See Table 4.

TABLE 4 Immune responses in C57BI/6 mice immunized two times  with tuberculosis lipopeptide based liposomal vaccines T cell IgG IgMproliferation IFN-g TB TB Vaccine (CPM) SI (pg/mL) dilipopep* dilipopep*TB Dilipo 35064 13.2 9769  1/218,700  1/900 TB Monolipo 19501 16.2 2276 1/24,000 <1/100 Saline   633  0.6  495 <1/100 <1/100 *TB dilipopeptideDQVHFQPLPPAVVKLSDALIK (SEQ ID NO: 2) was used as a solid phase in ELISAassay.

Two immunizations with dilipo-tuberculosis peptide increased the levelof IgG titers dramatically as compared to titers that were induced afterimmunizations with monolipo-tuberculosis peptide. The results indicatethat the presence of two lipids increases the titer that correlates to ahumoral response by almost 10 times.

Example 4 Mono- and Dilipidated Hepatitis B Peptides

TABLE 5 Protocol I368B Immune responses in mice immunized two timeswith hepatitis B mono- or dilipopeptide liposomal vaccine T cell IgG IgMproliferation IFN-g Hepatitis B Hepatitis B Vaccine (CPM) (pg/mL)dilipopep* dilipopep* Hepatitis B Dilipo 3243    0  1/48,600  1/2700Hepatitis B 5242 1034  1/100 <1/300 Monolipo Saline  158  361 <1/100<1/100 *Hepatitis B dilipopeptideIRTPPAYRPPNAPILK(Palmitate)K(Palmitate)G (SEQ ID NO: 10) was used as asolid phase in ELISA assay.

Two immunizations with dilipo-hepatitis B peptide increased dramaticallythe level of IgG titers, as compared to the titers that were inducedafter immunizations with monolipo-hepatitis B peptide. Similarly, thelevel of IgM antibodies is higher after dilipopeptide liposomeformulation is used for immunization procedure.

Example 5 Mono- and Dilipidated Malaria Peptides

TABLE 6 Protocol I368B Immune responses in mice immunized two timeswith malaria mono- or dilipopeptide liposomal vaccine T cell IgG IgMproliferation IFN-g Malaria Malaria Vaccine (CPM) (pg/mL) dilipopep*dilipopep* Malaria Dilipo 15075 1989  1/2700  1/2700 Malaria  1280 1036 1/100 <1/100 Monolipo Saline    74  361 <1/100 <1/100 *Malariadilipopeptide VTHESYQELVKKLEALEDAVK(Palmitate)K(Palmitate)G (SEQ ID NO:11) was used as a solid phase in ELISA assay.

Two immunizations with dilipo-malaria peptide increased the level ofboth IgG and IgM titers, as compared to the titers that were inducedafter immunizations with monolipo-malaria peptide. In this case malariadilipopeptide vaccine induced stronger cell proliferation and IFN-γlevels as compared to monolipo-vaccine. This shows that the intensity ofan immune response, i.e., the cellular immune response, can be modulatedby varying the number of lipids attached to the antigen.

Example 6 Varying the Ratio of Mono- and Dilipopeptides in a Liposome

By incorporating various ratios of mono- and dilipopeptides intoliposomes, it is possible to modulate the level and intensity of humoralresponses. Table 7 summarizes IgM titers in C57Bl/6 mice after twoimmunizations with various liposomal constructs. As expected, miceimmunized with BLP25 monolipopeptide or saline (group 5 and 6) did notproduce any detectable IgM titers. However, all liposomal formulationsgenerated a comparable cellular immune response (data not shown).

The mixture of monolipo- and dilipopeptides at 3:1 ratio, respectively,did not significantly improve antibody titers (group 1); however, thereis an increase in antibody titer, as compared to group 1 when mono- anddilipopeptides were incorporated into liposomes at 1:1 ratio. The mostintense antibody response was observed in the mice immunized with aliposomal formulation containing MUC1 monolipo- and dilipopeptides atthe ratio 1:3 (group 3).

Thus, by incorporating mono- and dilipopeptides at various ratios intoliposomes it is possible to modulate the level of humoral responses.

TABLE 7 Protocol 1399B (2× immunization) IgM Antibody  Titer Data IgMInjected C57B1/6 BP1-151-H S A Material Mouse Log2 Antibody×2 immunizations i.d. # Titer Titer Gr.1 BLP25 1  6.6  1/100Monolipo 300 μg/mL 2 <6.6 <1/100 Dilipo 100 μg/mL, 3  6.6  1/1002001 μg/mL 4  8.2  1/300 Lipid A 5 <6.6 <1/100 Gr.2 BLP25 1  6.6  1/100Monolipo 200 μg/mL 2  6.6  1/100 Dilipo 200 μg/mL, 3  8.2  1/300200- μg/mL 4  6.6  1/100 Lipid A 5  8.2  1/300 Gr.3 BLP25 1  9.8  1/900Monolipo 100 μg/mL 2 14.6  1/24,300 Dilipo 300 μg/mL, 3 11.4  1/2700200 μg/mL 4  9.8  1/900 Lipid A 5 13  1/8100 Gr.4 BLP25 1  9.8  1/900Dilipo 400 μg/mL 2  9.8  1/900 200 μg/mL 3 11.4  1/2700 Lipid A 4 11.4 1/2700 5  9.8  1/900 Gr.5 BLP25 1 <6.6 <1/100 Monolipo 400 μg/mL 2 <6.6<1/100 200 μg/mL 3 <6.6 <1/100 Lipid A 4  6.6  1/100 5 <6.6 <1/100 Gr.6Saline 1 <6.6 <1/100 2 <6.6 <1/100 3 <6.6 <1/100 4 <6.6 <1/100 5 <6.6<1/100 Monolipo [BP1-148 (SEQ ID NO: 12):STAPPAHGVTSAPDTRPAPGSTAPP-Lys(Pal)G] Dilipo [BP1-236 (SEQ ID NO: 13):STAPPAHGVTSAPDTRPAPGSTAPPK(Lipo)K(Lipo)G]

Example 7 The Effect of Lipid Chain Position in MUC1 Lipopeptide on theHumoral Immune Response

The demonstration of a potent antibody response after immunization ofmice with MUC1 lipopeptide with two liposerine residues attached to thecarboxy terminus raises a question if this strong antibody responsemight be maintained, if the position or number of liposerine residueswill be changed.

To answer this question several MUC1 lipopeptide constructs withdifferent liposerine residue placements were synthesized. The datapresented in Table 8 shows that only formulation #5, with threeliposerine residues at carboxy terminus, was able to generate a potentanti-MUC1 IgG response. The insertion of a three-serine spacer betweenthe two lipid chains (formulation #4) led to the decrease of the levelof antibody responses.

Similarly, formulation #2 where only one lipid chain was inserted in themiddle of the MUC1 peptide showed some low antibody titers, but thosetiters were much higher when two liposerine residues were inserted intothe central position of MUC1 molecule (formulation #1). Some lowantibody titers were observed when the liposerine residues were placedin extreme positions: one on the carboxy terminus and one on the aminoterminus (formulation #2).

The strongest antibody responses could be generated when two or threeliposerine residues are placed at carboxy terminus. Changing theposition of the liposerine residues still maintain some antibody titers,higher than BLP25 or saline (group 6 and 7) but significantly lower ascompared to carboxy terminus location.

TABLE 8 Protocol 1405 IgG Antibody titer data Injected BP1-265 MaterialMouse Log₂ Antibody # immunizations # Titer Titer Gr.1 Formulation #1 111.40  1/2700 (400 μg/mL BP1- 2 11.40  1/2700 271; 200 μg/mL lipid 3 9.80  1/900 A) × 2 4 13.00  1/8100 immunizations - i.d. 5 14.60 1/24,300 Gr.2 Formulation #2 1 <6.6 <1/100 (400 μg/mL BP1- 2 <6.6<1/100 273; 200 μg/mL lipid 3  8.20  1/300 A) × 2 4  8.20  1/300immunizations - i.d. 5  9.80  1/900 Gr.3 Formulation #3 1  9.80  1/900(400 μg/mL BP1- 2 <6.6 <1/100 272; 200 μg/mL lipid 3  8.20  1/300 A) × 24  8.20  1/300 immunizations - i.d. 5 <6.6 <1/100 Gr.4 Formulation #4 1 9.80  1/900 (400 μg/mL BP 1- 2  8.20  1/300 274; 200 μg/mL lipid 313.00  1/8100 A) × 2 4  6.60  1/100 immunizations - i.d. 5 11.40  1/2700Gr.5 Formulation #5 1 14.60  1/24,300 (400 μg/mL BP1- 2 16.20  1/72,900275; 200 μg/mL lipid 3 16.20  1/72,900 A) × 2 4 16.20  1/72,900immunizations - i.d. 5 16.20  1/72,900 Gr.6 Formulation #6 1 <6.6 <1/100(400 μg/mL BP1- 2 <6.6 <1/100 148; 200 μg/mL lipid 3 <6.6 <1/100 A) × 24 11.40  1/2700 immunizations - i.d. 5 <6.6 <1/100 Gr.7 Saline × 2 1<6.6 <1/100 immunizations - i.d. 2 <6.6 <1/100 3 <6.6 <1/100 4 <6.6<1/100 5 <6.6 <1/100 [BP1-255 (SEQ ID NO: 14):TSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVS(Lipo)S(Lipo)L] [BP1-271 (SEQID NO: 15): TSAPDTRPAPGSS(Lipo)S(Lipo)STSAPDTRPAPGS] [BP1-272 (SEQ IDNO: 16): TSAPDTRPAPGSS(Lipo)STSAPDTRPAPGS] [BP1-273 (SEQ ID NO: 17):S(Lipo)GVTSAPDTRPAPGSAS(Lipo)L] [BP1-274 (SEQ ID NO: 18):GVTSAPDTRPAPGSTAS(Lipo)SSSS(Lipo)L] [BP1-275 (SEQ ID NO: 19):GVTSAPDTRPAPGSTAS(Lipo)S(Lipo)S(Lipo)L] [BP1-148 (SEQ ID NO: 20):STAPDAHGVTSAPDTRPAPGSTAPP-Lys(Palmitoyl)]

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention. Thus, it is intended that the present invention cover themodifications and variations of this invention provided they come withinthe scope of the appended claims and their equivalents.

1.-81. (canceled)
 82. A method for inducing a humoral immune response,comprising administering to an individual a formulation comprising: atleast one liposome, which comprises a dilipopeptide, said dilipopeptidecomprising at least eleven contiguous amino acids of either SEQ ID NO:2,SEQ ID NO:3 or SEQ ID NO:4, and wherein two amino acids of saiddilipopeptide are lipidated.
 83. The method of claim 82 in which eachlipidated amino acid is selected from the group consisting of threonine,serine, lysine, arginine and cysteine.
 84. The method of claim 82wherein the lipidated amino acids are serine or lysine.
 85. The methodof claim 82 wherein two adjacent amino acids of the dilipopeptide arelipidated.
 86. The method of claim 82 in which each lipidated amino acidis lipidated with a lipid selected independently from the groupconsisting of myristyl, palmitoyl and lauryl.
 87. The method of claim 82wherein each lipidated amino acid is serine or lysine and each lipidatedamino acid is lipidated with myristyl or palmitoyl.
 88. The method ofclaim 82, which further comprises administering an adjuvant.
 89. Themethod of claim 88, wherein the adjuvant is selected from the groupconsisting of lipid A, monophosphoryl lipid A, muramyl dipeptide, andIL-2.
 90. The method of claim 88, wherein the adjuvant is lipid A ormonophosphoryl lipid A.
 91. The method of claim 82, which furthercomprises administering a monolipopeptide, wherein the monolipopeptidehas one lipidated amino acid and the identical sequence asdilipopeptide.
 92. The method of claim 82, wherein the dilipopeptideelicits a humoral IgG immune response which is detectably greater thanthe humoral IgG immune response elicited under the same conditions by anotherwise identical monolipopeptide wherein one of the two lipidatedamino acids of said dilipopeptide is unlipidated or is omitted.
 93. Themethod of claim 82, wherein the dilipopeptide comprises elevencontiguous amino acids of SEQ ID NO:2.
 94. The method of claim 82,wherein the dilipopeptide comprises SEQ ID NO:2.
 95. The method of claim82, wherein the dilipopeptide comprises eleven contiguous amino acids ofSEQ ID NO:3.
 96. The method of claim 82, wherein the dilipopeptidecomprises SEQ ID NO:3.
 97. The method of claim 82, wherein thedilipopeptide comprises eleven contiguous amino acids of SEQ ID NO:4.98. The method of claim 82, wherein the dilipopeptide comprises SEQ IDNO:4.
 99. A liposomal composition comprising a dilipopeptide, saiddilipopeptide comprising at least eleven contiguous amino acids ofeither SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, and wherein two aminoacids of said dilipopeptide are lipidated.
 100. The composition of claim99, wherein comprises eleven contiguous amino acids of SEQ ID NO:2. 101.The composition of claim 99, wherein comprises eleven contiguous aminoacids of SEQ ID NO:3.
 102. The composition of claim 99, whereincomprises eleven contiguous amino acids of SEQ ID NO:4.